Effect ofm-3m3FBS on Ca²⁺handling and viability in OC2 human oral cancer cells

Abstract

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 μM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.

Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

“…To deplete the ER-Ca 2+ stores, cells were perfused with the reversible Ca 2+ -ATPase (SERCA) inhibitor cyclopiazonic acid-CPA (20 μM, Sigma-Aldrich, USA), followed by the addition of mibefradil 100 μM in Ca 2+ -free Ringer's solution. To address the role of the PLC isoenzymes, cells were perfused with the PLC activator m-3M3FBS (60 μM) (Sigma-Aldrich, USA) as reported [47] followed by the addition of mibefradil 100 μM in regular extracellular Ca 2+ levels (2 mM Ca 2+ ). We also use the PLC blocker U73122 (5 μM) (Sigma-Aldrich, USA) simultaneously with the application of mibefradil (100 μM) [48].…”

U73122 and m-3M3FBS Regulate the GABAergic Neuron Regeneration via PLCβ in Planarian Dugesia japonica

“…m-3M3FBS treatment can overcome the anti-apoptotic effect of Bcl-2 or c-FLIPs in cancer cells [18]. m-3M3FBS also induces Ca 2+ -independent cell death and apoptosis [19,20]. This compound provides an opportunity to study PLC-mediated signaling.…”